There is an extensive literature relating to conjugates of biological significance which are deliberately designed to combine the activities of two previously independent substances. The most prominent example is enzyme linked immunoassay (ELISA), where an enzyme is conjugated to an antibody to serve as a label. Other applications include immunotoxins wherein a toxin is linked with an antibody to effect the specific delivery of the toxin to a target tissue, delivery of therapeutic agents by linking these agents to antibodies, and reagents for detection or localization of particular tissues by targeting specific tissue receptors with a heteromolecule containing a binding portion and label. A review of such conjugates in tumor therapy appeared in Immunological Reviews (1982) 62:1-216.
A number of approaches to formation of the desired linkages have been employed. Perhaps the most popular approach utilizes the commercially available reagent N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP). SPDP offers the possibility of disulfide bridge between itself and one moiety and an amide between itself and the other. Thus, for example, two proteins can be linked wherein one contains a free amino group with which to form the amide, and another contains a sulfhydryl to form the disulfide. Alternatively, each of two proteins with side chain amino groups can be derivatized with SPDP to provide the needed sulfhydryl functionality for a disulfide bridge between them.
Other available linkers react with protein native or created sulfhydryl groups by using for example a maleimido or haloacetyl group to form a thioether. These linkers generally have the functionality for an amide formation at the opposite end of the linking molecule by providing an activated carboxyl moiety. Thus for example, the activated esters of 6-maleimidocaproic acid, 2-bromoacetic acid, and 2-iodoacetic acid have been used, preferably forming the activated ester form of these acids by generation of the succinimidyl ester or the water soluble ester formed from 1-hydroxy-2-nitro-4-sulfonic acid sodium salt.
Other coupling agents that may be used include the more old fashioned and less specific linkers such as glutaraldehyde and dehydrating agents such as dicyclohexylcarbodiimide.
The foregoing linkers have been chosen with the thought that the two members of the conjugate are both proteins and the known side chain functionality of proteins has been capitalized upon. More recently, it has been realized that if a glycoprotein is one of the components, the sugar moieties of the saccharide portion of the glycoprotein could be employed. This has been suggested, for example, in European Patent Application publication no. 88695, published Sept. 14 1983. Specifically, among other suggestions, this published application discloses utilization of the carbohydrates present on glycoproteins in general and antibodies in particular by forming hydrazones with the oxidized form of the carbohydrate. The disclosure thus suggests reacting the aldehyde groups of an oxidized antibody saccharide component with a hydrazine reagent or an amine reagent. In that regard, the disclosure envisions using a bifunctional linker having a hydrazine or comparable moiety to the end of a bifunctional linker. No specific linkers containing a hydrazine moiety at one end and a group capable of reaction with sulfhydryl the other are suggested.
However such a specific linker is suggested for the purpose of binding label to cell surface carbohydrates by Taylor, K.E., et al, Biochemistry International (1980) 1:353-358 who suggest the use of 2-acetamido-mercaptobutyric acid hydrazide. This reagent provides an SH group capable of disulfide bond formation with a protein whose own sulfhydryl is activated by, for example, SPDP, and also provides a hydrazide for reaction with the oxidized carbohydrate.
It should be apparent that although there are in the art a number of heterobifunctional linking moieties (referred to by one commercial supplier of these as "double agents"), there is no specific linker class available which provides the capacity for reaction with an aldehyde at one of its termini and reaction to form a disulfide at the other. Such a class of reagents is particularly useful in binding glycoproteins to proteins which have or which can be provided with sulfhydryl groups.